New sorbent-based hydrophobic alginic acid derivatives for fat removal in multi-pesticide residues: analysis of a fatty food sample

Hydrophobic alginic acid derivatives were synthesized with various aliphatic hydrocarbon chains for fat removal in an analysis of multi-pesticide residues in a fatty food sample. First, alginic acid was chemically modified using eco-friendly ultrasound-assisted esterification with different alcohols, namely, hydrophobic alginic acid–methanol (HAA-C1), hydrophobic alginic acid–butanol (HAA-C4), and hydrophobic alginic acid–octadecanol (HAA-C18). The degree of esterification (DE) was determined by titration, and the results ranged from 57.3% to 63.7%. The physicochemical properties of the synthesized hydrophobic alginic acids (HAAs) were studied using FT-IR, XRD, TGA, and FE-SEM. Subsequently, the performance of the HAAs was checked and evaluated for the removal of fat from a fatty food sample by calculating the fat removal percentage and the determination of 214 pesticide residues in the fatty food sample. For the fat removal percentage application, the HAAs were able to efficiently remove between 77% and 83% of the fat; HAA-C18 had the highest percentage. Regarding the pesticide residue application, HAAs were also able to remove the fat content from the fatty food sample without a significant effect on the pesticide substances. The recoveries of the detected pesticide compounds were between 80% and 120% for all HAAs. However, there were various missing pesticide compounds for HAAs. The number of missing pesticide compounds was 19, 6, and 33 for HAA-C1, HAA-C4, and HAA-C18, respectively. HAA-C4 had medium hydrophobicity and it lost fewer pesticides than the other HAAs. This was because the multi-pesticide mixture had various classes of chemical structure; hence, it had different polarity powers. We concluded that HAAs are developable and applicable to be safely used as a green material in diverse fatty food sample analysis applications.


Introduction
Alginic acid is a heteropolysaccharide linear copolymer consisting of b-D-mannuronic acid (M) and a-L-guluronic acid (G) residues, which are connected through (1,4) glycosidic bonds and are arranged in either repeating GG (MM) blocks or alternating MG structures.Alginic acid is mainly derived from brown seaweed; it has been widely studied and used in different biomedical elds due to its remarkable characteristics, including its low cost, non-toxicity, superior biodegradability, and biocompatibility 1 , 2 .Despite the proven interest, alginic acid use remains limited due to the abundance of carboxyl and hydroxyl groups on the molecular chains.These dramatically increase the formation of intramolecular hydrogen bonds in alginate chains, making the structure rigid and stretched.The resulting difficult-to-load hydrophobic materials possess poor compatibility toward hydrophobic compounds.An abundance of hydrophilic groups decreases the stability of alginic acid in biological buffers, exhibiting unexpected and uncontrollable kinetic degradation and extensive water uptake properties 3,4 .
Recently, researchers have focused on the development of hydrophobic materials from alginates through covalent modi-cations; this eld of research is rapidly growing.One approach involves the esterication of carboxyl groups by incorporating long-chain alkyl groups or aromatic rings.Alginates are then esteried using carbodiimide derivatives as a coupling reagent. 1 Benzoyl chloride was used to substitute the hydroxy group with the benzoyl group. 2 It is also possible to obtain alginate esters by treating tetrabutylammonium salts of alginic acid with alkyl halides.Alginates have been modied by direct esterication through Fischer esterication with selected alcohols in the presence of a suitable catalyst. 5However, the limited use of the Fischer esterication reaction is related to the degradation of the polysaccharide chain in the presence of strongly acidic conditions and high temperatures within the long-time reaction necessary to obtain a high degree of conversion.Research has been developed to overcome this limitation by decreasing the reaction temperature.Broderick et al. investigated the possibility of obtaining alginate butyl esters by applying sodium alginate, butanol, and H 2 SO 4 as a catalyst at room temperature for 18 h. 6Murdzheva exploited ultrasonic irradiation to obtain methylated, ethylated, and isopropylated derivatives of alginic acid.Their nding revealed that an ultrasound-assisted synthesis performed at 45 kHz reduced the reaction time to 2 hours in contrast to esterication performed under conventional conditions of heating. 7here are many studies focused on the synthesis of lipophilic esters using polysaccharide biopolymers to enhance their properties and increase their potential for use in various applications. 8Chitin, cellulose, and agarose are other types of polysaccharide biopolymers that have been developed to obtain lipophilic esters.In chitin, chitin acyl ester compounds were synthesized with short and long chains of fatty acids to distinguish emulsifying, 9 crystallinity, and thermal properties. 10egarding the cellulose and cellulose derivatives, the modication by esterication process has taken place to produce lipophilic cellulose ester. 11In a previous study, carboxymethyl cellulose (CMC) was modied using various hydrocarbon chains by esterication via an eco-friendly ultrasound-assisted procedure to remove fat content in food analysis applications. 12In addition, cellulose ester was modied with extracted fatty acids from sunower oil to be used as an additive in resins for vat photopolymerization. 13 Agarose fatty acid esters are another modied biopolymer produced by the esterication process and are used as hydrocolloid surfactants to investigate their emulsifying ability. 14Also, agarose was modied using gallic acid to be used as coated lms for seafood preservation in food packaging. 15esticides are multiple; various chemical groups have the ability to kill, repel, or mitigate animals, insects, or cultivated plant pests. 16Although pesticides save and protect crops and agricultural production worldwide, they pose a critical threat to human health when they are misused and can be transferred to the human body by consuming foodstuff. 17Therefore, there have been global collaborative efforts to limit the spread and misuse of pesticides; many regulations have been set for this purpose and monitoring methods have been increased/ developed, whether via professional laboratories or farm inspections.Pesticides including insecticides, herbicides, and fungicides are classied into four main groups, based on their chemical composition, namely, organophosphate, organochlorine, pyrethroid, and carbamate. 18Their diversity in structure consequently affects the polarity strength of each group based on the attached function groups.
One of the most challenging issues in the detection of pesticides in foodstuff is the fat content in fatty food samples.Fat has several negative effects on the extraction process of pesticides as well as on the analytical instruments used, including contamination, carryover, and the blockage of columns or pipes. 19Many well-known techniques have been used to remove the fat from fatty food samples, for instance, liquid-liquid extraction with a strong non-polar solvent, Soxhlet extraction, microwave-assisted extraction, and freezing precipitation. 20Despite these attempts, there remains a requirement for a simple, low-cost, and efficient method to effectively perform the task.The QuEChERS (quick, easy, cheap, effective, rugged, and safe) method is a recent and widely used method used to extract pesticide residue from foodstuff, 21 but it does not measure the fat content and is not able to clean the sample extractor of the fat content. 22herefore, we aimed to synthesize a hydrophobic alginic acid to be used as a sorbent clean-up material to be then used in the pesticide residue detection of a fatty food sample.Initially, the chemical modication of alginic acid was conducted using ecofriendly ultrasound-assisted esterication with different alcoholic hydrocarbon chains.Subsequently, the efficiency of the fabricated material was evaluated by calculating the fat removal percent and by analyzing a real animal product sample, which was spiked with pesticide substances.

Synthesis of hydrophobic alginic acid (HAA)
Alginic acid was modied using environmentally friendly ultrasound-assisted esterication with three different alcohols (methanol, butanol, and octadecanol).The synthesis of HAAs was adopted from previous work 7,23 .The reaction took place under heterogeneous conditions.Briey, three conical asks with caps were prepared, then 1 g alginic acid (AA) was weighed into each one.Next, 200 mL methanol was added to rst sample (HAA-C1), 450 mL butanol was added to the second sample (HAA-C4), and 4.5 g octadecanol and 200 mL methanol as medium (1 : 1 mole) were added to the third sample (HAA-C18).The molar ratio of AA and all alcohols was 1 : 1. Subsequently, 1 mL sulfuric acid at a concentration of 96% was added to each sample, then all samples were placed in a constant ultrasonic water bath for 2 h at 40 °C.Finally, each new synthesized HAA was ltered then washed twice using 70% ethanol, followed by acetone.The new products were then dried in an oven at 40 °C for 1 h.The degree of esterication (DE) was determined by titration, as illustrated in the Food Chemical Codex. 24The physical and chemical properties of the synthesized biopolymers were checked using characterization tools (FT-IR, XRD, TGA, and SEM).

Determination of fat% removal
To investigate the performance of the synthesized hydrophobic AAs from a fat removal aspect, 2 g of chicken (a fat sample) was weighed in a 50 mL centrifuge tube, and then 10 mL of acetonitrile was added.Aer shaking for 2 minutes and centrifuging at 4000 rpm for 5 minutes at room temperature, the liquid layer (acetonitrile) was transferred to another tube and evaporated under the N 2 evaporation system until dryness.The remaining fat matrix was weighed and recorded, then dissolved again using 10 mL of acetonitrile.At this stage, the HAAs were added, shaken for 10 minutes, and centrifuged again in the same condition as mentioned above to precipitate the HAAs with fat matrix content.Aer that, the acetonitrile layer was moved to a new tube and then evaporated until dryness, and the remaining fat was weighed and recorded again.The fat% removal was calculated by dividing the wt fat aer using HAAs over the wt.fat before, then multiplying by 100.This process was repeated three times, and the average percentage was reported for each HAA.

Determination of pesticide residues in a fat sample using HAAs
The sample preparation and the extraction of pesticide residues from a fat sample were adopted from an international method to minimize the error sources. 25Briey, 2 g of a homogenized chicken sample was weighed in a 50 mL centrifuge tube and spiked with 30 mg kg −1 of a mixed pesticide standard.Subsequently, 10 mL acetonitrile was added and shaken for 2 min.The extraction salt (QuEChERS) was then added to the mixture and it was manually shaken for 2 min, then centrifuged at 4000 rpm for 5 min at room temperature.Next, 1 mL of the extractor (upper layer) was transferred to a new 2 mL Eppendorf tube.HAA biopolymers were added to remove the fat content.The mixture was shaken for 5 min, then centrifuged again for 5 min at 4000 rpm at 4 °C.Finally, the upper layer was moved to an HPLC vial and injected into LC-MS/MS.
The analysis was performed using a liquid chromatographymass spectrometer 6500 triple quad instrument (±ESI-LC-MS/ MS) and a 1290 Innity UPLC system with ideal conditions.
The analytical column used was reverse phase (C18; 2.1 × 150 mm; 1.8 mm).The mobile phase consisted of water; each had a 5 mM ammonium format with 1% formic acid.The MS parameters were set for optimized conditions, with an ion source temperature of 550 °C, ion spray potential of 5500 V, and input potential of 10 V.The quantication of the measured pesticides was calculated using a solvent calibration curve (external calibration strategy).

Chemistry
The modied ester of alginic acid HAAs was successfully synthesized using a simple, fast, cost-effective, and eco-friendly method by combining acid catalyzed with ultrasound-assisted irradiation.Methyl, butyl, and octadecyl alginates were esteri-ed, as shown in Scheme 1.The heterogeneous synthesis of HAAs in the (methanol/H + ) depended on the surface interaction between the solid phase (alginic acid) and the liquid medium (methanol/H + ).The use of ultrasound in the irradiation process signicantly reduced the reaction time from 72 h to just 2 h. 7he ultrasound also generated gas microbubbles that implosively disrupted, forming cavitation bubbles at incredibly high temperatures and pressures.These actions resulted in a significant acceleration of the reaction.In HAA-C18, methanol was used as a medium because the octadecanol is in powder form as well as alginic acid, so there is a possibility of esterication with methanol instead of octadecanol.In spite of that, octadecanol competed with methanol in this interaction due to its higher molecular weight.FT-IR spectroscopy supported this interpretation.Thereaer, the degree of esterication of AA was determined and provided at 63.7 ± 4.6%, 57.3 ± 2.7%, and 59.4 ± 3.1% for HAA-C1, HAA-C4, and HAA-C18, respectively.

Characterization conrmation
3.2.1 FT-IR analysis.The successful synthesis of HAAs was veried by FT-IR spectra, as shown in Fig. 1.The FT-IR spectra of HAA-C1, HAA-C4, and HAA-C18 were compared with the AA spectrum reported in a previous work. 26HAA-C1, HAA-C4, and HAA-C18 exhibited additional weak bands apart from the basic characteristic absorption bands of AA.The absorption bands of HAA-C1 and HAA-C4 at 2930, 2927, and 2926 cm −1 signicantly enhanced HAA-C18, representing the stretching vibration of -CH 2 on the methyl, butyl, and octadecyl groups.Extra bands appeared at 1734, 1733, and 1740 cm −1 , owing to the stretching vibration of C]O on the ester groups.The other new signals that appeared at 1245, 1243, and 1244 cm −1 were attributed to the stretching vibration of C-O on the ester group.These results indicated that the methyl, butyl, and octadecyl groups had successfully graed onto alginate via the esterication reaction to generate the HAAs.
3.2.2X-ray diffraction (XRD) analysis.XRD is the most direct and effective method to analyze the change of crystal structures of alginates in esterication reactions.As shown in Fig. 2, HAA-C1, HAA-C4, and HAA-C18 exhibited weak crystalline diffraction peaks, indicating their amorphous structures.
Diffraction peaks of AA at 13.4°and 22.7°are typical characteristic peaks of hydrated crystalline structures, resulting from the intermolecular hydrogen bonds of AA. 27 HAA-C1, HAA-C4, and HAA-C18 revealed a sharp diffraction peak at 2q = 14.8°a nd a wider diffraction peak at 2q = 20°aer the chemical modication, similar to the previously reported structure characteristics of alginate derivatives. 3][30] 3.2.3Thermal gravimetric analysis (TGA).A thermogravimetric analysis was used to estimate the different thermal stabilities of alginic acid as well as the aliphatic ester derivatives.The measurement has been carried out at a heating rate of 10°per minute under an argon atmosphere.The mass loss curves of HAA-C1, HAA-C4, and HAA-C18 are presented in Fig. 3.
From the thermograms, we observed that all three materials exhibited different thermal behaviors, indirectly reecting the change in molecular structures.The TGA of the alginic acid AA curve was reported as three consecutive weight-loss steps.The rst stage of weight loss, at approximately 16.05% of the initial weight, was probably due to intermolecular water molecules from the polymer. 31The IDT abbreviation represents the initial degradation temperature at which the decomposition starts.All the measured materials showed nearly similar IDT values (195-200 °C), which are higher values compared to the pure AA sample.Fig. 3 demonstrates that the weight losses of the rst stage of HAA-C1, HAA-C4, and HAA-C18 were approximately 13%, 9%, and 11%, respectively.Similar degradation behavior was observed for the IDT results, as illustrated in Table 1.This phenomenon was a direct effect of the hydrophobic nature of the introduced aliphatic groups, which caused a reduction of intermolecular water in the alginic acid. 2 Scheme 1 Preparation mechanism of the modified hydrophobic alginic acids HAA-C1, HAA-C4, and HAA-C18.
The second weight loss occurred at an inection point of 200 °C.This degradation step was the main step (fastest step) for all modied derivatives (HAA-C1, HAA-C4, and HAA-C18).This behavior designated the decomposition of the alginate by dehydrating the saccharide rings, breaking the C-H and C-O-C glycoside bonds in the main polysaccharide chain.The data in Table 1 demonstrate the thermal behavior of the modied structure, where 10%, 20%, 30%, and 40% represent the weight loss.No signicant change between AA and HAA-C1 was observed at 10%; at 20% to 40%, the degradation decreased with an increase in temperature as the side chain of ester rose.HAA-C4 and HAA-C18 modied with a exible side chain showed steady state thermal stability started at 450 and 300 °C, respectively, compared with AA and HAA-C1, which had fewer thermal stability characteristics.
Finally, the thermograms showed a highly thermal stable structure in the third degradation step of HAA-C18, caused by the decrease in the carboxyl groups of the polymer and the formation of ester bonds that destroyed the polymeric intramolecular hydrogen bond.All the outcomes conrmed that the esterication of alginic acid was successfully introduced.The nal degradation temperature (FDT) refers to the temperature at which the decomposition is completed or becomes nal.The literature conrms that pure AA is completely decomposed at 700 °C. 31Whereas, the other materials show signicantly higher FDT values in comparison to pure AA.The FDT values are in the following order: HAA-C1 = HAA-C4 < HAA-C18.Such observations are attributed to the measurement conditions under an argon atmosphere, which reduce the thermal degradation process.
3.2.4Morphological analysis.The TGA study of HAA-C18 was found to have high thermal stability over a range of temperatures.To investigate this stability, the morphologies of HAA-C18 were studied using scanning electron microscopy (SEM) at various magnications.From the micrographs (shown in Fig. 4), the surface was less folded and the folding pattern    roughness displayed a porous surface with a network of crevices and protrusions, observed more at higher magnications.At lower magnications (Fig. 4(a and b)), the surface morphology appeared to comprise irregular semi-spheroids with uneven surfaces and microscopic pores.At higher magnications (Fig. 4(c and d)), the surface exhibited short and irregular bers, with an approximate width of 0.25 mm.At extremely high magnications of approximately 120 K (Fig. 4(e and f)), the surface showed an aggregation of short, at, and non-uniform strips, approximately 92.59 to 38.48 nm.Regular patterns have been reported from alginic acid as a folded surface structure; 2 hence, all the changes seen in the obtained morphology conrmed the esterication of alginic acid.The modications resulted from a shi in the internal order, which inuenced the polymer crystal structure with heterogeneous molecules between the polymeric alginic acid chains and the immobilization of the aliphatic chain with glucose hydroxyl groups on the repeating polysaccharide units.

Fat% removal application
The physical appearance of the nal extractor was an initial indicator of HAAs working; it became clearer and ner aer applying HAAs in the extraction procedure.HAAs were able to remove approximately 77-83% of the fat in the clean-up step.HAA-C18 had the highest removal percent of approximately 83%; the lowest was achieved by HAA-C4.Table 2 shows the fat% removal of each HAA.In general, all HAAs were able to  Fig. 5 The chromatograms of peak shapes for a few pesticide compounds.Paper RSC Advances efficiently remove fat and the results were close to each other.Therefore, the results were accepted for the intended purpose, and are promising for future developments.

Pesticide residue application
The ability of the fabricated HAAs to remove fat from the sample was checked using HAAs as a clean-up in a pesticide residue Fig. 6 The recoveries of pesticide compounds after using HAAs.Fig. 7 The number of missing pesticide compounds after using HAAs.
application of a fatty sample.Pesticide compounds were chosen due to their diversity of chemical structures; they have wide groups of semi-polar and non-polar substances.Therefore, this method was appropriate to investigate the performance of HAAs to rend off the fat without affecting the extraction and recovery of analytes.An LC mixture standard containing 214 compounds from various chemical pesticide classes such as organochlorine, organophosphorous, carbamate, and pyrethroid was used and spiked in the fatty animal product sample.First, pesticide compounds were analyzed without the HAAs to check their stability and activity.Subsequently, HAAs were applied as a clean-up step and the results were evaluated.Fig. 5 shows the integrated peak shapes for a few of the representative pesticide compounds.
The effect of HAA-C1, HAA-C4, and HAA-C18 on the pesticide compounds was individually evaluated; the analyte recoveries are reported in Table 3.All HAAs provided satisfactory recoveries for the detected pesticide compounds.The recovery ranged from 80% to 120%, which met the requirement speci-cations according to SANTE/2017. 23Fig. 6 illustrates the recovery ranges for the pesticide compounds aer applying the three HAAs.There were various missing pesticides aer using HAAs as a fat sorbent.The number of missing pesticide compounds were 19, 6, and 33 for HAA-C1, HAA-C4, and HAA-C18, respectively, as displayed in the pie chart in Fig. 7.The un-unied missing pesticide compounds among HAA sorbents could be interpreted as being due to the difference in the chemical interactions between pesticides and HAAs.The highest number of missing compounds appeared aer using HAA-C18, where 33 pesticides were lost (including 4,4 ′ -dichlorobenzophenon, cycloxydim, and carbaryl).Aer checking the structure of the missed pesticide compounds, it was clearly observed that they had high hydrophobicity properties that could chemically interact with the most hydrophobic HAA-C18; hence, they were probably removed with the fat in the clean-up stage.HAA-C1 and HAA-C4 which had short hydrocarbon chains and, hence, fewer hydrophobicity properties-lost fewer pesticide compounds.It was observed that HAA-C1 lost a greater number of compounds than HAA-C4 (e.g., ufenoxuron), possibly due to the high polarity.In conclusion, HAAs could be used as an efficient removal sorbent for fat in pesticide residue applications, but we highly recommend that the various structures of the wide groups of pesticide compounds and the consequential effects are considered.
It is crucial to mention that HAAs should not be renewed or reused since they react with the food sample's fat matrix and will precipitate and be discarded at the conclusion of the analysis.Rewashing or eliminating the fat content with an organic solvent may cause structural damage to HAAs or eliminate it along with fat molecules.

Conclusion
In this project, alginic acid was chemically modied using an eco-friendly esterication procedure with three alcohols (methanol, butanol, and octadecanol) to obtain hydrophobic alginic acids (HAAs).The synthesis of HAAs occurred and was conrmed by FT-IR, TGA, XRD, and SEM.Subsequently, the performance of these biopolymers was evaluated by measuring their ability to remove the fat content of a fatty food sample.The fat removal percent was calculated for each HAA.The highest percent was recorded for HAA-C18, but all materials were successful, with approximate values between 77% and 83%.The fabricated HAAs were used in a real fatty food sample analysis to determine multi-pesticide residues.All HAAs were satisfactorily able to remove fat from a nal extractor, and pesticide compounds were successfully recovered.HAA-C4 provided fewer missing multi-class pesticide compounds compared with HAA-C1 and HAA-C18 due to its medium hydrophobicity.Therefore, HAAs are developable and a promising and efficient method to be used in food analysis applications.

Table 2
The fat% removal of HAAs biopolymers

Table 3
The result of recovery study for pesticide compounds after using HAAs © 2024 The Author(s).Published by the Royal Society of Chemistry RSC Adv., 2024, 14, 2491-2503 | 2497 Paper RSC Advances